Surgery is the most common treatment for all types of solid tumors. A successful cancer surgery is to completely remove cancer cells and maximally preserve normal structures. To improve cancer surgery outcomes, fluorescent molecular probes have been developed and increasingly used for guiding tumor removal during surgery. Fluorescence emitted from these intraoperative molecular probes enables surgeons to visualize tumor tissues in real time and perform fluorescence-guided tumor resection. It has been well demonstrated that fluorescence-guided tumor resection enables more complete tumor resection and enhances treatment outcomes. Aminolevulinic acid (ALA) is one of a few FDA-approved intraoperative fluorescent probes. Although ALA itself is not fluorescent, it is metabolized in the heme biosynthesis pathway to a fluorescent and photosensitizing metabolite protoporphyrin IX (PpIX), enabling fluorescence detection and photodynamic therapy (PDT). ALA-PpIX has been clinically used for guiding the resection of various types of tumors. However, clinical applications of ALA are limited by issues such as low PpIX tumor production, PpIX fluorescence heterogeneity, and low tumor/normal fluorescence contrast. Our goal is to optimize ALA-based modalities by addressing tumor phenotypic and genotypic characteristics that affect tumor PpIX fluorescence. Two optimization strategies are proposed in this research. Specific Aim 1 is to optimize ALA-PpIX fluorescence-guided tumor resection and treatment by suppressing PpIX efflux. We have found that triple negative breast cancer (TNBC) cell lines show reduced ALA-PpIX fluorescence and are resistant to ALA-PDT due to elevated ABCG2 transporter activity. We have searched for clinical ABCG2 transporter inhibitors and will use them to improve ALA-PpIX fluorescence-guided resection and treatment of TNBC tumors with increased ABCG2 transporter activity. Specific Aim 2 is to optimize the dose of ALA for PpIX fluorescence-guided resection and treatment of tumors with genetic alterations in heme biosynthesis enzymes. We have found that pancreatic cancers exhibit genetic alterations in heme biosynthesis enzymes, which change ALA-PpIX production profile. We will optimize ALA dose to increase PpIX fluorescence contrast between tumor and normal tissues to improve fluorescence-guided resection of pancreatic cancers. Through this research, we hope to demonstrate that ALA protocols optimized to fit tumor phenotype and genotype offer better surgical outcomes than applying it based on one-size-fits-all approach.